EP3275995B1 - Streptomyces et procédé pour la production de la milbémycine a4 au moyen dudit streptomyces - Google Patents

Streptomyces et procédé pour la production de la milbémycine a4 au moyen dudit streptomyces Download PDF

Info

Publication number
EP3275995B1
EP3275995B1 EP16771322.1A EP16771322A EP3275995B1 EP 3275995 B1 EP3275995 B1 EP 3275995B1 EP 16771322 A EP16771322 A EP 16771322A EP 3275995 B1 EP3275995 B1 EP 3275995B1
Authority
EP
European Patent Office
Prior art keywords
milbemycin
streptomyces
fermentation
powder
medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP16771322.1A
Other languages
German (de)
English (en)
Other versions
EP3275995A4 (fr
EP3275995A1 (fr
Inventor
Yun TENG
Meidong XU
Meiyi MO
Zhihui HE
Zhengjie Chen
Lianqing JIANG
Hua Bai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang Hisun Pharmaceutical Co Ltd
Original Assignee
Zhejiang Hisun Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang Hisun Pharmaceutical Co Ltd filed Critical Zhejiang Hisun Pharmaceutical Co Ltd
Publication of EP3275995A1 publication Critical patent/EP3275995A1/fr
Publication of EP3275995A4 publication Critical patent/EP3275995A4/fr
Application granted granted Critical
Publication of EP3275995B1 publication Critical patent/EP3275995B1/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces
    • C12R2001/55—Streptomyces hygroscopicus

Definitions

  • the present invention relates to a new streptomyces and a method for producing milbemycin A4 by the fermentation culture of the streptomyces.
  • Milbemycin is natural product from microbial source, which can be used as pesticide, and there have been data showing that it is one of the best acaricides in the modern world.
  • the US Environmental Protection Agency identifies it as a low-risk pesticide and Holland approves it as "GNO" (natural product in the production of crops). It belongs to ecology friendly pesticide and is applicable to the comprehensive prevention and control of organic agriculture pests and diseases, which has become a popular insecticidal and acaricidal agent in developed countries.
  • milbeknock milbeknock
  • milbemycin emulsifiable concentrate In 1993, 1% of milbemycin emulsifiable concentrate was also registered in Japan as a pesticide for pears, peaches, watermelons, strawberries, eggplants and flowers.
  • milbemycin has been registered in many countries, such as Japan and many countries in Europe and America, etc. and is recommended by the US Environmental Protection Agency to be used as a safe, environment friendly insecticidal and acaricidal agent.
  • the unit content of milbemycin A3+milbemycin A4 reached a maximum of 1595mg/L in the literature published in 2011 ( Zhang Baoxin et al, African J Biotechnol 10(37):7225-7235 ), and the unit content of milbemycin A3+ milbemycin A4 increased to 2237 mg/L in the Chinese patent CN 103468625 A .
  • the fermentation broth containing milbemycin has complex composition, wherein milbemycin A3 and milbemycin A4 are main components and other structural analogues are further comprised.
  • the ratio of milbemycin A3 to milbemycin A4 in the fermentation broth containing milbemycin is about 1:2, so the existing commercial product is also a mixture of components of A3 and A4.
  • the main reason leading to this result is that it is difficult to separate A3 and A4 single component from the fermentation broth with similar proportions of A3 and A4, which affects the final yield.
  • Achieving the single component production of A4 can further facilitate the application of milbemycin A4 single component in other fields.
  • the most effective way to obtain a single component is to obtain a new strain capable of producing the single component and to further increase the fermentation unit.
  • nsdA was cloned and sequenced, which presented an 89.89% identity with that of S. coelicolor.
  • the lambdaRED-mediated PCR-targeting technique was used to create nsdA replacement in the S. bingchengensis _226541 chromosome.
  • the nsdA disruption mutant, BC29 was obtained, which produced more pigment and spores than did the ancestral strain.
  • HPLC analysis revealed that the disruption of nsdA efficiently increased milbemycin A(4) production and nanchangmycin production by 1.5-fold and 9-fold, respectively. Complementation of the nsdA mutation restored the phenotype and antibiotic production. These results showed that nsdA negatively affected sporulation and antibiotic production in S. bingchengensis.
  • CN 103 789 339 A relates to the technical field of gene recombination, and particularly relates to streptomycete generating 5-keto-milbemycins and a method for producing 5-keto-milbemycins.
  • the invention provides a recombinant vector for knocking out the milF gene of streptomycete, including the homologous fragment of the milF gene, wherein the sequence of the fragment is obtained by losing 105-767 nucleotides from the nucleotides from the sites 158-924 in the nucleotide sequence shown by SEQ ID No:2.
  • the invention also provides an establishment method of the recombinant vector, a method for knocking out the milF gene in streptomycete by use of the recombinant vector and streptomycete without milF gene.
  • the streptomycete can be directly fermented to generate 5-keto-milbemycins, so that the synthesis process of milbemycin oxime is simplified, and the pollution caused by the traditional chemical synthesis method is avoided.
  • an object of the present invention is to provide a microbial strain capable of increasing the unit yield of milbemycin A4, which is characterized in that the content of milbemycin A4 in its fermentation broth accounts for a percentage of more than 80% of the total content of A3 and A4 and the unit yield of milbemycin A4 can reach greater than 4000ug/ml, the unit yield of milbemycin A3+milbemycin A4 can reach greater than 5000ug/ml, and the fermentation unit is high and the impurity content is low.
  • the microbial strain of Streptomyces HS7522 of the present invention was deposited on September 16, 2014 at China General Microbiological Culture Collection Center (Address: Institute of Microbiology, Chinese Academy of Sciences, No.1 Beichen West Road, Chaoyang District, Beijing) with an accession number of CGMCC No. 9671, classified and nominated as Streptomyces hygroscopicus, registered and proved to be survival.
  • the main biological characteristics of Streptomyces HS7522 of the present invention are as follows: the colonies on ISP1 medium are white, on ISP2 medium pebble grey and on ISP3 medium are bright ivory; rounded, slightly raised in the middle, rugose and medium size of diameter (about 6mm or so); except for on ISP1 medium, the spores abundant, substrate mycelia developed, mycelia are closely combined with medium and not easy to provoke; no pigment is produced on ISP1 and ISP2 media and khaki grey pigment is produced on ISP3 medium.
  • the present invention describes the characteristics of Streptomyces HS7522 on morphology and molecular levels. It can be confirmed that Streptomyces HS7522 belongs to Streptomyces hygroscopicus by comparing with known milbemycin-producing bacteria on the morphology and molecular levels. Streptomyces HS7522 has 99.5% homology with Streptomyces sp. NRRL 5739 16s rRNA and 99.9% homology with Streptomyces bingchenggensis BCW-1. The biggest difference between this strain and most of the milbemycin-producing bacteria in morphology is that the colonies of other strains, such as Streptomyces sp. CGMCC No. 7677, will secrete golden tears on the colony surface on the production plate, however, HS7522 on the same medium is stale grey and has no tears on the surface (see Figure 2 ).
  • the present invention further provides a method for preparing milbemycin by using Streptomyces HS7522 (CGMCC No. 9671).
  • the method comprises the process of aerobic fermentation of Streptomyces HS7522 (CGMCC No. 9671) in nutrient medium containing assimilable carbon source and assimilable nitrogen source.
  • the above assimilable carbon source is preferably selected from one of starch, dextrin, glucose, industrial molasses, glycerol, sucrose, lactose, maltose, trehalose, xylan, mannitol and sorbitol or a combination of the above substances.
  • the above assimilable nitrogen source is preferably selected from one of yeast extract, yeast powder, beef extract, tryptone, peptone, skim milk powder, whole milk powder, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn pulp dry powder, bran, urea and ammonium salt or a combination of the above substances.
  • the nutrient medium contains 2-12 g/L yeast extract, yeast extract paste or peptone, 20-200 g/L sucrose or molasses, 2-11 g/L skim milk powder or corn pulp, 5-11 g/L soybean cake powder, 5-15 g/L cottonseed cake powder or gluten powder, 0.2-1 g/L K 2 HPO 4 , 0.05-0.1 g/L FeSO 4 • 7H 2 O, 0.005-0.02 g/L ZnSO 4 , 1-5 g/L CaCO 3 , 0.01-0.05 g/L CuSO 4 and /or 0.1-0.5 g/L Na 2 MoO 4 .
  • the temperature of the fermentation culture is preferably 20-40°C, preferably 25-35°C
  • the pH of the medium is 6.0-8.0, preferably about 7.0
  • the culture time is 300-360 h
  • the dissolved oxygen is not less than 35%
  • the ventilation volume is 0.5-1.0vvm.
  • the fermentation mode is submerged fermentation.
  • Milbemycin can be detected by the following HPLC method:
  • the milbemycin-producing strain adopted in the present invention is Streptomyces HS7522 (CGMCC No. 9671), a spontaneous mutant or a mutant obtained by conventional mutagenesis of Streptomyces HS7522 (CGMCC No. 9671).
  • Sucrose is the product of Guangxi Dongmen Nanhua Sugar Industry Co., Ltd.
  • Yeast extract is the product of Zhejiang Dongcheng Pharmaceutical Co., Ltd.
  • Yeast extract paste is the product of Hefei Laisi Biological Engineering Co., Ltd.
  • Peptone is the product of Huzhou Confluence Biological science and technology Co., Ltd.
  • Molasses is the product of Guangdong Jiangmen Biological science and technology Co., Ltd.
  • Skim milk powder is the product of Hulunbeier Sanyuan Milk Co.,Ltd.
  • Corn pulp is the product of Shandong Shouguang Juneng Golden Corn Co., Ltd.
  • Soybean cake powder is the product of Ningbo Beilun Jiangnan Oil Co., Ltd.
  • Cottonseed cake powder is the product of Beijing Kang Mingwei Medium Technology Co., Ltd.
  • Gluten powder is the product of Beijing Kang Mingwei Medium Technology Co., Ltd.
  • the Streptomyces hygroscopicus HS7522 of the present invention is a strain with high proportion of milbemycin A4, which is obtained by multiple rounds of mutagenesis and selective breeding (including the mutagenic means, such as NTG, EMS, UV, etc.) based on the milbemycin producing strain Streptomyces milbemycinicus CGMCC No. 7677 (see the Chinese Patent CN103789339A ) which was deposited by the applicant of the present application. Compared with the original strain, the appearance of the strain has changed greatly, which belongs to morphological mutant.
  • Streptomyces milbemycinicus CGMCC No.7677 strain was cultured on ISP3 slant medium at 28°C for 10-12 d. Next, its mycelia were scraped under sterile conditions with an inoculating shovel, ground in a ground mouth tube and then suspended in sterile water to obtain bacterial suspension. The bacterial suspension was subjected to mutagenesis using NTG (nitrosoguanidine), EMS (ethylmethane sulfonate), UV (ultraviolet).
  • EMS 1 ml of EMS was taken and dissolved in 2 ml of absolute ethyl alcohol and 22ml of 0.1 mol/L phosphate buffer (pH 7.2) was further added.
  • 5ml of single-cell bacterial suspension with 10 -1 or 10 -2 gradient was added to a plate equipped with a clip.
  • 5ml of 4.0% EMS solution was then added into the plate and the final concentration of EMS solution was 2.0%.
  • the plate was placed on the magnetic stirrer and stirred for 20-60 min.
  • 10ml of 5% sodium thiosulfate was added into the mutagenesis plate to stop the reaction.
  • the resulting mixture was then diluted for 10 2 -10 7 times with physiological saline in turn and the dilutions were coated on ISP3 plates respectively to obtain mutagenesis groups.
  • the colors and pigments of mycelia were observed after cultivation at 28°C for 7-10 d on 10 kinds of media, i.e. ISP1, ISP2, ISP3, ISP4, ISP5, Gause's No.1, calcium malate, nutrient agar, YMS and Czapek's (culture characteristics were shown in Table 1).
  • the mycelia of the present invention grown well on ISP2 were collected and inoculated in TSB liquid medium containing glass beads, placed in an incubator at 28°C and shake cultured at 250 r/min for 2-4 d. Then the mycelia were collected by centrifugation and washed twice with sterile water and then stored at 4°C for use.
  • the reaction was carried out on a PCR cycler.
  • the procedure was as follows: pre-denaturation at 95°C for 5min, 30 cycles of denaturation (94°C for 45s), annealing (55°C for 45s) and extension (72°C for 90s), then extension at 72°C for 10min.
  • the reaction system is as follows: deionized water 14.25 ⁇ L 10 ⁇ PCR Buffer 2.0 ⁇ L dNTP mixture 0.5 ⁇ L Taq polymerase 0.25 ⁇ L Primer 1(27F) 1.0 ⁇ L Primer 2(1495R) 1.0 ⁇ L DNA template 1.0 ⁇ L (quantified according to concentration)
  • the PCR products were detected by 0.8% agarose gel electrophoresis. The products of clear bands were selected to be purified. The amplified products were recovered by gel electrophoresis and ligated to the T vector for sequencing. The sequence of the primary structure of 16S rDNA of the strain was obtained. The results showed that Streptomyces HS7522 had 99.5% homology with Streptomyces sp. NRRL 5739 16s rRNA and 99.9% homology with Streptomyces bingchenggensis BCW-1 by performing similarity search in the Genebank database (blast). Table 8 Homologies between Streptomyces HS7522 and related strains Species name GenBank No.
  • Streptomyces HS7522 of the present invention its aerial mycelia on the SP2 medium were pebble grey, the back was beige red and there was no tear on the colony surface and no pigment was produced; its aerial mycelia on ISP4 medium were ivory, the back was ivory and there was no tear on the colony surface, no pigment was produced and arabinose and xylose cannot be utilized.
  • Streptomyces HS7522 of the present invention belongs to genus Streptomyces, but it is different from the known milbemycin-producing strain Streptomyces NRRL NO. 5739 and Streptomyces bingchengsis sp.nov CGMCC No. 1734. Streptomyces HS7522 is a new strain.
  • slant spore medium yeast extract 2, malt extract 2, sucrose 10, skim milk powder 1.2, agar 20, pH was 7.0-7.2 before sterilization.
  • Test tube was 30 ⁇ 200 mm and filled volume was 20 mL. After sterilization at 121°C for 20 min, the medium was cooled to 50-60°C to form slant. The slant was then inoculated with a ring of mycelia. After cultivation at the temperature of 28+1 °C for 10 d, the mycelia were mature.
  • seed medium g/L: yeast extract 5, peptone 5, sucrose 20, skim milk powder 3, K 2 HPO 4 0.8, pH was 6.8-7.2 before sterilization.
  • the filled volume of shaking flask was 250mL and triangular flask 30mL.
  • the seed medium was sterilized at 121°C for 20 min.
  • the inoculation amount of the bacteria was 10 5 -10 6 c.f.u./mL, the culture temperature was 28 ⁇ 1°C, and shaking cultured at 250 rpm for 48 h in shaker.
  • fermentation medium g/L: yeast extract 5, sucrose 100, skim milk powder 11, soybean cake powder 10, cottonseed cake powder 14, K 2 HPO 4 1, FeSO 4 • 7H 2 O 0.1, ZnSO 4 0.02, CaCO 3 5, CuSO 4 0.05, Na 2 MoO 4 0.5.
  • the filled volume of shaking flask was 250mL and triangular flask 30mL.
  • the fermentation medium was sterilized at 121°C for 20 min and then inoculated with seed culture in an inoculation amount of 10% (volume percentage) and shaking cultured at the temperature of 28+1 °C, 250 rpm for 14 d in shaker. After fermentation, the fermentation broth was detected by HPLC.
  • the HPLC detection method of milbemycin is as follows: 0.5 ml of fermentation broth was taken and 4.5 ml of 75% ethanol was then added and mixed homogenously. The resulting mixture was centrifuged at 3000 rpm for 15 min and the supernatant was taken for sample injection.
  • HPLC mobile phase The conditions of HPLC mobile phase were as follows: Table 9 Gradient Elution Flow rate (ml/min) Time (min) Acetonitrile Methanol Water 1.0 0 50 24 26 1.0 6 50 24 26 1.0 10 53 24 23 1.0 12 53 24 23 1.0 15 60 20 20 1.0 16 60 20 20 1.0 17 100 0 0 2.0 18.5 100 0 0 2.0 18.6 50 24 26 1.0 21 50 24 26 Injection volume: 10 ⁇ l.
  • the target products in the fermentation broth were determined to be milbemycin A3 and milbemycin A4 (the target products were determined to be milbemycin A3 and milbemycin A4 by comparison of HPLC chromatograms of the fermentation brothes of the following examples).
  • the content of milbemycin A4 in the fermentation broth was 3900mg/L, the content of A3 was 967 mg/L, and the content of milbemycin A4 accounted for 80.1% of the total content of A3 and A4.
  • the formulation of fermentation medium was the same as that in Example 5, except that 0.25% of defoamer needed to be added as antifoaming agent.
  • the volume of fermentor was 50L and the volume of feed material was 35L.
  • the pH of the fermentation medium was 7.0-7.6 before sterilization. Steam sterilization was then carried out at 121°C for 25 min. After cooling, about 3.5L of seeding tank culture was inoculated therein and then fermented at the temperature of 28 ⁇ 1°C, stirring rake stirred with the lowest rate of 150rpm depending on the dissolved oxygen, which was not less than 35%.
  • the ventilation volume was 0.6vvm for 14 d and then the fermentation broth in the fermentor was released.
  • the fermentation broth was detected by HPLC as shown in Example 5, and in the fermentation broth, the content of milbemycin A4 was determined to be 4100 mg/L, the content of A3 was 962 mg/L, and the content of milbemycin A4 accounted for 81% of the total content of A3 and A4.
  • 8T of seed medium (see Example 5 for the formulation of seed medium, meanwhile 0.25% of defoamer was added as antifoaming agent) was fed into 15T seeding tank. Steam sterilization was carried out at 121°C for 35 min. After the sterilization, the volume of the medium was 10T. After cooling, 2 L of shake flask seed culture was inoculated therein and then cultured at the temperature of 28 ⁇ 1°C, stirring rate of 100rpm and ventilation volume of 0.8wwm for 48 h.
  • the formulation of fermentation medium was the same as that in Example 5, except that 0.3% of defoamer was added as antifoaming agent.
  • the fermentor was 70T and the volume of feed material was 55T.
  • the pH was 6.8-7.6 before sterilization. Steam sterilization was carried out at 121°C for 35 min. After cooling, about 6T of seeding tank culture was inoculated therein and then fermented at the temperature of 28 ⁇ 1°C, stirring rake stirred with the lowest rate of 50rpm depending on the dissolved oxygen, which was not less than 35%.
  • the ventilation volume was 0.5vvm for 14 d and then the fermentation broth in the fermentor was released.
  • the fermentation broth was detected by HPLC as shown in Example 5, and in the fermentation broth, the content of milbemycin A4 was determined to be 4020 mg/L, the content of A3 was 961 mg/L, the content of milbemycin A4 accounted for 80.7% of the total content of A3 and A4.
  • seed medium g/L: yeast extract powder 5, peptone 5, sucrose 40, K 2 HPO 4 0.5, pH was 7.0-7.4 before sterilization.
  • the filled volume of shaking flask was 250mL and triangular flask 25 mL.
  • the seed medium was sterilized at 121 °C for 20 min.
  • a piece of bacteria clump with an area of 1 ⁇ 2 cm was dug from the slant in Example 5 and inoculated into a seed bottle and cultured at 28+1 °C for 45 hr.
  • the fermentation broth was determined by HPLC as shown in Example 5, and in the fermentation broth, the content of milbemycin A4 was determined to be 3960 mg/L, the content of A3 was 929 mg/L, the content of milbemycin A4 accounted for 81% of the total content of A3 and A4.
  • Seed culture was prepared according to Example 8 and then 2.5 mL of seed culture was inoculated into the fermentation medium (g/L): peptone 10, sucrose 140, corn pulp 10, soybean cake powder 10, gluten powder 15, K 2 HPO 4 1, FeSO 4 • 7H 2 O 0.1, ZnSO 4 0.02, CaCO 3 5, CuSO 4 0.05, Na 2 MoO 4 0.5, with the filled volume of shaking flask 250mL and triangular flask 30mL, which were sterilized at 121°C for 20 min. The resulting mixture was then shaking cultured at 28+1 °C, 250 rpm in shaker for 14 d.
  • the fermentation medium g/L
  • the fermentation broth was determined by HPLC as shown in Example 5, and in the fermentation broth, the content of milbemycin A4 was determined to be 4100 mg/L, the content of A3 was 900 mg/L, the content of milbemycin A4 accounted for 82% of the total content of A3 and A4.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Claims (9)

  1. Streptomyces hygroscopicus HS7522, qui est déposée sous le numéro d'accès N° 9671 du CGMCC et possède la capacité de produire de la milbémycine.
  2. Utilisation de Streptomyces HS7522 selon la revendication 1 pour la production de milbémycine.
  3. Procédé de fermentation de milbémycine, comprenant l'étape de fermentation aérobie de Streptomyces HS7522, qui est déposée sous le numéro d'accès N° 9671 du CGMCC, dans un milieu de nutriments contenant une source de carbone assimilable et une source d'azote assimilable.
  4. Procédé selon la revendication 3, dans lequel la source de carbone assimilable est sélectionnée parmi l'une de l'amidon, la dextrine, le glucose, des mélasses industrielles, le glycérol, le saccharose, le lactose, le maltose, le tréhalose, le xylane, le mannitol et le sorbitol, ou une combinaison des substances ci-dessus.
  5. Procédé selon la revendication 3, dans lequel la source d'azote assimilable est sélectionnée parmi l'une d'un extrait de levure, une poudre de levure, un extrait de viande de bœuf, la tryptone, la peptone, une poudre de tourteau de soja, une poudre de tourteau de coton, une poudre de tourteau d'arachide, une poudre de gluten, une poudre sèche de pulpe de maïs, des fibres, l'urée et un sel d'ammonium, ou une combinaison des substances ci-dessus.
  6. Procédé selon l'une quelconque des revendications 3 à 5, dans lequel le milieu de nutriments contient 2-12 g/l d'extrait de levure, de pâte d'extrait de levure ou de peptone, 20-200 g/l de saccharose ou de mélasses, 2-11 g/l de poudre de lait écrémé ou de pulpe de maïs, 5-11 g/l de poudre de tourteau de soja, 5-15 g/l de poudre de tourteau de coton ou de poudre de gluten, 0,2-1 g/l de K2HPO4, 0,05-0,1 g/l de FeSO4·7H2O, 0,005-0,02 g/l de ZnSO4, 1-5 g/l de CaCO3, 0,01-0,05 g/l de CuSO4 et/ou 0,1-0,5 g/l de Na2MoO4.
  7. Procédé selon la revendication 3, dans lequel la température de la fermentation est de 20-40 °C, de préférence 25-35 °C ; le pH du milieu est de 6,0-8,0, de préférence 7,0 ; le temps de fermentation est de 300-360 h.
  8. Procédé selon l'une quelconque des revendications 3 à 5, dans lequel l'oxygène dissous est au minimum de 35 % pendant la fermentation.
  9. Procédé selon l'une quelconque des revendications 3 à 5, dans lequel le volume de ventilation est de 0,5-1,0 vvm pendant la fermentation.
EP16771322.1A 2015-03-27 2016-03-25 Streptomyces et procédé pour la production de la milbémycine a4 au moyen dudit streptomyces Active EP3275995B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510137374.2A CN106148215B (zh) 2015-03-27 2015-03-27 一种链霉菌及其生产米尔贝霉素a4的方法
PCT/CN2016/077355 WO2016155567A1 (fr) 2015-03-27 2016-03-25 Streptomyces et procédé pour la production de la milbémycine a4 au moyen dudit streptomyces

Publications (3)

Publication Number Publication Date
EP3275995A1 EP3275995A1 (fr) 2018-01-31
EP3275995A4 EP3275995A4 (fr) 2018-09-19
EP3275995B1 true EP3275995B1 (fr) 2020-03-04

Family

ID=57003865

Family Applications (1)

Application Number Title Priority Date Filing Date
EP16771322.1A Active EP3275995B1 (fr) 2015-03-27 2016-03-25 Streptomyces et procédé pour la production de la milbémycine a4 au moyen dudit streptomyces

Country Status (5)

Country Link
US (1) US10526668B2 (fr)
EP (1) EP3275995B1 (fr)
JP (1) JP6467521B2 (fr)
CN (1) CN106148215B (fr)
WO (1) WO2016155567A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106148216B (zh) 2015-03-27 2019-06-04 浙江海正药业股份有限公司 一种链霉菌及其生产米尔贝霉素a3的方法
CN109022516A (zh) * 2018-05-31 2018-12-18 四川大学 一种提高米尔贝链霉菌抗生素产量的方法
CN109810925B (zh) * 2019-03-18 2023-04-18 陕西麦可罗生物科技有限公司 一种改进的多抗霉素发酵培养基及发酵工艺
CN109971692A (zh) * 2019-05-16 2019-07-05 华东理工大学 林可链霉菌（Streptomyces lincolnensis）及培养方法和应用
CN111607547B (zh) * 2020-05-06 2021-10-22 中国农业科学院植物保护研究所 一种碳源吸收表达系统、重组菌及应用
CN113588840A (zh) * 2021-08-17 2021-11-02 丽珠集团福州福兴医药有限公司 一种检测米尔贝霉素含量的方法
CN114195840B (zh) * 2021-12-20 2024-02-27 台州科技职业学院 一种大环内酯类化合物结构鉴定的方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3950360A (en) 1972-06-08 1976-04-13 Sankyo Company Limited Antibiotic substances
AR243528A1 (es) * 1986-12-11 1993-08-31 Sankyo Co Procedimiento para preparar compuestos de macrolida y un procedimiento para producir con los mismos una composicion parasiticida.
JPH0889272A (ja) * 1994-09-29 1996-04-09 Mitsubishi Chem Corp ミルベマイシン系化合物及びその製造法
CN101100651B (zh) * 2007-05-28 2010-05-26 东北农业大学 链霉菌属菌株及其应用方法
CN103468625B (zh) * 2013-09-10 2015-05-13 东北农业大学 一种冰城链霉菌的基因阻断突变菌及其制备方法和应用
CN103789339B (zh) * 2013-11-04 2017-08-04 浙江海正药业股份有限公司 一种产5‑酮基米尔贝霉素的链霉菌及生产5‑酮基米尔贝霉素的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
JP6467521B2 (ja) 2019-02-13
US10526668B2 (en) 2020-01-07
CN106148215A (zh) 2016-11-23
JP2018509166A (ja) 2018-04-05
US20190048427A1 (en) 2019-02-14
EP3275995A4 (fr) 2018-09-19
EP3275995A1 (fr) 2018-01-31
WO2016155567A1 (fr) 2016-10-06
CN106148215B (zh) 2019-06-14

Similar Documents

Publication Publication Date Title
EP3275995B1 (fr) Streptomyces et procédé pour la production de la milbémycine a4 au moyen dudit streptomyces
EP3275996B1 (fr) Streptomyces et procédé de production de milbémycine a3 à l'aide de celui-ci
CN108676757B (zh) 一株链霉菌属菌株及其产星孢菌素的应用
US20190284521A1 (en) A bacillus subtilis strain, culture method and use thereof
CN112300957B (zh) 一种喜盐芽孢杆菌及其工业化生产依克多因的方法
US9982314B2 (en) Bacterial strain of Actinoplanes sp. and application thereof
CN102061278B (zh) 一种食甲基菌mp688及其应用
CN113999794A (zh) 一株链霉菌及其应用
Chen et al. Enhancement and selective production of avermectin B by recombinants of Streptomyces avermitilis via intraspecific protoplast fusion
CN117467571A (zh) 一种降解烟嘧磺隆的芽孢杆菌及应用
CN105176904B (zh) 基因工程菌株筑波链霉菌l21及其应用
WO2023016387A1 (fr) Bacillus amyloliquefaciens et son utilisation dans la préparation de 1-désoxynojirimycine
CN107815426B (zh) 发酵生产春雷霉素的专用菌株及其应用
CN108410759B (zh) 一株高效降解氯氟氰菊酯的玫瑰色红球菌及其应用
EP0313297B1 (fr) Procédé de production d'aglycones d'avermectine et des cultures les contenant
CN108410760B (zh) 一株高效降解氰戊菊酯的酯香微杆菌及其应用
CN106190854A (zh) 一种荒漠拟孢囊菌和奥利万星中间体的制备方法
Bailey et al. Use of mutagenic agents in improvement of α‐amylase production by bacillus subtilis
CN110423790A (zh) 一种抗真菌四霉素b定向高产的代谢工程方法
CN117568237A (zh) 一种格尔德霉素链霉菌及其发酵生产格尔德霉素的应用
Roth et al. Use of chemostat for selection of Streptomyces hygroscopicus mutants altered in regulation of maltose utilization
CN117757869A (zh) 一种高效发酵生产格尔德霉素的方法
Zhu et al. Isolation and identification of the antagonistic strain DM-54 of Bacillus amyloliquefacien against Verticillium dahliae, and optimization of antifungal protein producing conditions
CN114854613A (zh) 一种雪白链霉菌菌株及其用途和生产新生霉素的方法
CN114891656A (zh) 一种枯草芽孢杆菌及其在发酵产腺苷中的应用

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20170921

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20180821

RIC1 Information provided on ipc code assigned before grant

Ipc: C12P 17/18 20060101ALI20180814BHEP

Ipc: C12R 1/55 20060101ALN20180814BHEP

Ipc: C12N 1/20 20060101AFI20180814BHEP

RIC1 Information provided on ipc code assigned before grant

Ipc: C12R 1/55 20060101ALN20190705BHEP

Ipc: C12N 1/20 20060101AFI20190705BHEP

Ipc: C12P 17/18 20060101ALI20190705BHEP

RIC1 Information provided on ipc code assigned before grant

Ipc: C12R 1/55 20060101ALN20190712BHEP

Ipc: C12P 17/18 20060101ALI20190712BHEP

Ipc: C12N 1/20 20060101AFI20190712BHEP

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 1/20 20060101AFI20190902BHEP

Ipc: C12P 17/18 20060101ALI20190902BHEP

Ipc: C12R 1/55 20060101ALN20190902BHEP

INTG Intention to grant announced

Effective date: 20190917

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1240373

Country of ref document: AT

Kind code of ref document: T

Effective date: 20200315

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602016031130

Country of ref document: DE

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200604

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20200304

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200604

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200605

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200704

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200729

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1240373

Country of ref document: AT

Kind code of ref document: T

Effective date: 20200304

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602016031130

Country of ref document: DE

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20200331

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200325

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200331

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200325

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200331

26N No opposition filed

Effective date: 20201207

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20200331

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20200304

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230522

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20240307

Year of fee payment: 9

Ref country code: GB

Payment date: 20240325

Year of fee payment: 9

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20240325

Year of fee payment: 9